Compositions and methods for diagnosis or prognosis of testicular cancer

ABSTRACT

Provided are compositions and methods for diagnosis or prognosis of testicular or male germ-cell derived cancer, comprising: obtaining sperm DNA from a test subject; determining the methylation status of at least one CpG dinucleotide sequence of at least one gene sequence selected from HRAS, NTF3, MT1A, PAX8, DIRAS3, PLAGL1, SFN, SAT2CHRM1, MEST, RNR1, CYP27B1 and ICAM1; and thereby determining or diagnosing testicular or male germ-cell derived cancer. Provided are compositions and methods for identifying agents that cause testicular or male germ-cell derived cancer, comprising: obtaining human ES-cell derived primordial germ cells; contacting the germ cells or descendants thereof, with a test agent; culturing the contacted cells; determining, using a genomic DNA of the sample, the methylation status of at least one CpG dinucleotide sequence of at least one gene sequence selected from the above group; and identifying at least one test agent that causes testicular or male germ-cell derived cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 61/110,890, filed 3 Nov. 2008 and entitled COMPOSITIONS AND METHODS FOR DIAGNOSIS OR PROGNOSIS OF TESTICULAR CANCER, which is incorporated herein by reference in its entirety.

FEDERAL FUNDING ACKNOWLEDGEMENT

This work was at least in part supported by the Southern California Environmental Health Sciences Center Grant No. 5P30ES007048, funded by the National Institute of Environmental Health Sciences. The United States government therefore has certain rights in the invention.

FIELD OF THE INVENTION

Particular aspects relate generally to DNA methylation and epigenetic reprogramming during development, gametogenesis and cancer, and more particularly to novel and effective epigenetic biomarkers and methods for diagnosis or prognosis of testicular or male germ-cell derived cancer, comprising determining the methylation status of at least one CpG dinucleotide sequence of at least one gene sequence selected from HRAS, NTF3, MT1A, PAX8, DIRAS3, PLAGL1, SFN, SAT2CHRM1, MEST, RNR1, CYP27B1 and ICAM1. Additional aspects relate to compositions and methods for identifying and/or screening for agents that cause testicular or male germ-cell derived cancer, comprising contacting human (or murine, rat, Etc.) ES-cell derived primordial germ cells with a test agent and determining the methylation status of at least one CpG dinucleotide sequence from at least one sequence as disclosed herein.

BACKGROUND

Ten to twenty percent of couples attempting pregnancy are infertile. Male-factor infertility accounts entirely for approximately 20% of these cases, and is contributory in an additional 30% [1,2]. Well defined causes of male-factor infertility are known to include congenital and acquired dysfunction of the hypothalamic-pituitary-testicular endocrine axis, anatomic defects, chromosomal abnormalities, and point mutations [3-5]. However, these diagnoses account for only a small proportion of cases, and etiology remains unknown for most male-factor infertility patients [1,2].

The mammalian germ line undergoes extensive epigenetic reprogramming during development and gametogenesis. In males, dramatic chromatin remodeling occurs during spermatogenesis [6,7], and widespread erasure of DNA methylation followed by de novo DNA methylation occurs developmentally in two broad waves [6, 8-11]. The first occurs before emergence of the germ line, establishing a pattern of somatic-like DNA hypermethylation in cells of the pre-implantation embryo that are destined to give rise to all cells of the body, including germ cells. The second widespread occurrence of erasure takes place uniquely in primordial germ cells. Subsequent de novo methylation occurs during germ cell maturation and spermatogenesis, establishing a male germ line pattern of DNA methylation that remains hypomethylated compared with somatic cell DNA [8, 12-16].

A small number of studies have addressed the epigenetic state of the human male germ line. Substantial variation in DNA methylation profiles is reported in ejaculated sperm of young, apparently healthy men. Notable distinctions were observed both between samples from separate men and among individually assayed sperm from the same man [17].

Although this variation suggests that DNA methylation may be used as a biomarker of sperm quality, semen quality and fertility were not assessed in this study [17].

SUMMARY OF EXEMPLARY ASPECTS

Male-factor infertility is a common condition, and etiology is unknown for a high proportion of cases. Abnormal epigenetic programming of the germline is disclosed as a mechanism compromising spermatogenesis of some men currently diagnosed with idiopathic infertility. During germ cell maturation and gametogenesis, cells of the germ line undergo extensive epigenetic reprogramming. This process involves widespread erasure of somatic-like patterns of DNA methylation followed by establishment of sex-specific patterns by de novo DNA methylation.

According to particular aspects, incomplete reprogramming of the male germ line results in both altered sperm DNA methylation and compromised spermatogenesis.

Particular aspects provide the first discovery and disclosure ever of a broad epigenetic defect associated with abnormal semen parameters. Additional aspects relate to an underlying mechanism for these broad epigenetic changes, comprising improper erasure of DNA methylation during epigenetic reprogramming of the male germ line.

Concentration, motility and morphology of sperm was determined in semen samples collected by male members of couples attending an infertility clinic. METHYLIGHT™ and ILLUMINA™ assays were used to measure methylation of DNA isolated from purified sperm from the same samples. Methylation at numerous sequences was elevated in DNA from poor quality sperm, and provide novel and effective epigenetic biomarkers of sperm quality, semen quality and fertility.

Particular exemplary aspects, provide methods for determining or diagnosing abnormal sperm or fertility, comprising: obtaining a sample of human sperm DNA from a test subject; determining, using the genomic DNA of the sample, the methylation status of at least one CpG dinucleotide sequence of at least one gene sequence selected from the group consisting of HRAS, NTF3, MT1A, PAX8, DIRAS3, PLAGL1, SFN, SAT2CHRM1, MEST, RNR1, CYP27B1 and ICAM1; and determining, based on the methylation status of the at least one CpG sequence, the presence or diagnosis of abnormal sperm or fertility with respect to the test subject. In certain aspects, the determined methylation status of the at least one CpG sequence is hypermethylation. In particular embodiments, determining the methylation status of at least one CpG dinucleotide sequence comprises treating the genomic DNA, or a fragment thereof, with one or more reagents to convert 5-position unmethylated cytosine bases to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties. Preferably, treating comprises use of bisulfite treatment of the DNA.

In certain aspects, the at least one gene sequence is selected from the group consisting of HRAS SEQ ID NOS:63 and 20, NTF3 SEQ ID NOS:2 and 14, MT1A SEQ ID NOS:4 and 16, PAX8 SEQ ID NOS:1 and 13, DIRAS3 SEQ ID NOS:3 and 15, PLAGL1 SEQ ID NOS:7 and 19, SFN SEQ ID NOS:6 and 18, SAT2CHRM1 SEQ ID NOS:9 and 21, MEST SEQ ID NOS:5 and 17, RNR1 SEQ ID NOS:10 and 22, CYP27B1 SEQ ID NOS:11 and 23 and ICAM1 SEQ ID NOS:12 and 24.

In particular aspects, abnormal sperm comprises at least one of abnormal sperm concentration, abnormal motility, abnormal total normal morphology, abnormal volume, and abnormal viscosity. In certain embodiments, abnormal sperm comprises at least one of abnormal sperm concentration, abnormal motility, and abnormal total normal morphology.

Certain aspects of the methods, comprise determining, using the genomic DNA of the sample, the methylation status of at least one CpG dinucleotide sequence of at least one gene sequence selected from the group consisting of HRAS, NTF3, MT1A, PAX8 and PLAGL1. In certain embodiments, the at least one gene sequence is selected from the group consisting of HRAS SEQ ID NOS:63 and 20, NTF3 SEQ ID NOS:2 and 14, MT1A SEQ ID NOS:4 and 16, PAX8 SEQ ID NOS:1 and 13, and PLAGL1 SEQ ID NOS:7 and 19.

Yet additional aspects, provide methods for determining or diagnosing abnormal sperm or fertility, comprising: obtaining a sample of human sperm DNA from a test subject; determining, using the genomic DNA of the sample, the methylation status of at least one CpG dinucleotide sequence of at least one gene sequence from each of a repetitive DNA element sequence group, a maternally imprinted gene sequence group, and a non-imprinted gene sequence group; and determining, based on the methylation status of the at least one CpG sequence from each of the groups, the presence or diagnosis of abnormal sperm or fertility with respect to the test subject. In certain implementations, the at least one gene sequence from a repetitive element group comprises at least one selected from the group consisting of SAT2CHRM1 SEQ ID NOS:9 and 21. In certain aspects, the at least one gene sequence from a maternally imprinted gene group comprises at least one selected from the group consisting of PLAGL1 SEQ ID NOS:7 and 19, MEST SEQ ID NOS:5 and 17, and DIRAS3 SEQ ID NOS:3 and 15. In particular embodiments, the at least one gene sequence from a non-imprinted gene group comprises at least one selected from the group consisting of HRAS SEQ ID NOS:63 and 20, NTF3 SEQ ID NOS:2 and 14, MT1A SEQ ID NOS:4 and 16, PAX8 SEQ ID NOS:1 and 13, SFN SEQ ID NOS:6 and 18, RNR1 SEQ ID NOS:10 and 22, CYP27B1 SEQ ID NOS:11 and 23 and ICAM1 SEQ ID NOS:12 and 24.

Yet further aspects provide methods for screening for agents that cause spermatogenic deficits, abnormal sperm or abnormal fertility comprising: obtaining human ES-cell derived primordial germ cells; contacting the germ cells or descendants thereof, with at least one test agent; culturing the contacted germ cells or the descendants thereof under conditions suitable for germ cell proliferation or development; obtaining a sample of genomic DNA from the contacted cultured germ cells or the descendants thereof; determining, using the genomic DNA of the sample, the methylation status of at least one CpG dinucleotide sequence of at least one gene sequence selected from the group consisting of HRAS, NTF3, MT1A, PAX8, DIRAS3, PLAGL1, SFN, SAT2CHRM1, MEST, RNR1, CYP27B1 and ICAM1; and identifying, based on the methylation status of the at least one CpG sequence, at least one test agent that causes at least one of spermatogenic deficits, abnormal sperm, and abnormal fertility. In certain aspects, the determined methylation status of the at least one CpG sequence is hypermethylation. In certain embodiments, the at least one gene sequence is selected from the group consisting of HRAS SEQ ID NOS:63 and 20, NTF3 SEQ ID NOS:2 and 14, MT1A SEQ ID NOS:4 and 16, PAX8 SEQ ID NOS:1 and 13, DIRAS3 SEQ ID NOS:3 and 15, PLAGL1 SEQ ID NOS:7 and 19, SFN SEQ ID NOS:6 and 18, SAT2CHRM1 SEQ ID NOS:9 and 21, MEST SEQ ID NOS:5 and 17, RNR1 SEQ ID NOS:10 and 22, CYP27B1 SEQ ID NOS:11 and 23 and ICAM1 SEQ ID NOS:12 and 24.

Additional aspects provide methods for diagnosis or prognosis of testicular or male germ-cell derived cancer, comprising: obtaining a sample of human sperm DNA from a test subject; determining, using the genomic DNA of the sample, the methylation status of at least one CpG dinucleotide sequence of at least one gene sequence selected from the group consisting of HRAS, NTF3, MT1A, PAX8, DIRAS3, PLAGL1, SFN, SAT2CHRM1, MEST, RNR1, CYP27B1 and ICAM1; and determining, based on the methylation status of the at least one CpG sequence, diagnosis or prognosis of testicular or male germ-cell derived cancer with respect to the test subject. In certain aspects, the determined methylation status of the at least one CpG sequence is hypermethylation. In particular aspects, determining the methylation status of at least one CpG dinucleotide sequence comprises treating the genomic DNA, or a fragment thereof, with one or more reagents to convert 5-position unmethylated cytosine bases to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties. In certain embodiments, treating comprises use of bisulfite treatment of the DNA. In particular aspects, the at least one gene sequence is selected from the group consisting of HRAS SEQ ID NOS:63 and 20, NTF3 SEQ ID NOS:2 and 14, MT1A SEQ ID NOS:4 and 16, PAX8 SEQ ID NOS:1 and 13, DIRAS3 SEQ ID NOS:3 and 15, PLAGL1 SEQ ID NOS:7 and 19, SFN SEQ ID NOS:6 and 18, SAT2CHRM1 SEQ ID NOS:9 and 21, MEST SEQ ID NOS:5 and 17, RNR1 SEQ ID NOS:10 and 22, CYP27B1 SEQ ID NOS:11 and 23 and ICAM1 SEQ ID NOS:12 and 24.

In certain aspects, the diagnosis or prognosis is of germ-cell derived testicular cancer.

Particular embodiments of the cancer diagnostic or prognostic assays comprise determining, using the genomic DNA of the sample, the methylation status of at least one CpG dinucleotide sequence of at least one gene sequence selected from the group consisting of HRAS, NTF3, MT1A, PAX8 and PLAGL1. In certain aspects, the at least one gene sequence is selected from the group consisting of HRAS SEQ ID NOS:63 and 20, NTF3 SEQ ID NOS:2 and 14, MT1A SEQ ID NOS:4 and 16, PAX8 SEQ ID NOS:1 and 13, and PLAGL1 SEQ ID NOS:7 and 19.'

Additional aspects provide a method for diagnosis or prognosis of testicular or male germ-cell derived cancer, comprising: obtaining a sample of human sperm DNA from a test subject; determining, using the genomic DNA of the sample, the methylation status of at least one CpG dinucleotide sequence of at least one gene sequence from each of a repetitive DNA element sequence group, a maternally imprinted gene sequence group, and a non-imprinted gene sequence group; and determining, based on the methylation status of the at least one CpG sequence from each of the groups, diagnosis or prognosis of testicular or male germ-cell derived cancer with respect to the test subject. In certain aspects, the determined methylation status of the at least one CpG sequence is hypermethylation. In particular implementations, the methylation status of at least one CpG dinucleotide sequence comprises treating the genomic DNA, or a fragment thereof, with one or more reagents to convert 5-position unmethylated cytosine bases to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties. In certain embodiments, treating comprises use of bisulfite treatment of the DNA. In certain aspects, the at least one gene sequence from a repetitive element group comprises at least one selected from the group consisting of SAT2CHRM1 SEQ ID NOS:9 and 21. In particular aspects, the at least one gene sequence from a maternally imprinted gene group comprises at least one selected from the group consisting of PLAGL1 SEQ ID NOS:7 and 19, MEST SEQ ID NOS:5 and 17, and DIRAS3 SEQ ID NOS:3 and 15. In certain embodiments, the at least one gene sequence from a non-imprinted gene group comprises at least one selected from the group consisting of HRAS SEQ ID NOS:63 and 20, NTF3 SEQ ID NOS:2 and 14, MT1A SEQ ID NOS:4 and 16, PAX8 SEQ ID NOS:1 and 13, SFN SEQ ID NOS:6 and 18, RNR1 SEQ ID NOS:10 and 22, CYP27B1 SEQ ID NOS:11 and 23 and ICAM1 SEQ ID NOS:12 and 24.

Additionally provided are methods for screening for agents that cause testicular or male germ-cell derived cancer, comprising: obtaining human ES-cell derived primordial germ cells; contacting the germ cells or descendants thereof, with at least one test agent; culturing the contacted germ cells or the descendants thereof under conditions suitable for germ cell proliferation or development; obtaining a sample of genomic DNA from the contacted cultured germ cells or the descendants thereof; determining, using the genomic DNA of the sample, the methylation status of at least one CpG dinucleotide sequence of at least one gene sequence selected from the group consisting of HRAS, NTF3, MT1A, PAX8, DIRAS3, PLAGL1, SFN, SAT2CHRM1, MEST, RNR1, CYP27B1 and ICAM1; and identifying, based on the methylation status of the at least one CpG sequence, at least one test agent that causes testicular or male germ-cell derived cancer. In certain aspects, the determined methylation status of the at least one CpG sequence is hypermethylation. In certain aspectgs, determining the methylation status of at least one CpG dinucleotide sequence comprises treating the genomic DNA, or a fragment thereof, with one or more reagents to convert 5-position unmethylated cytosine bases to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties. In certain embodiments, treating comprises use of bisulfite treatment of the DNA. In particular embodiments, the at least one gene sequence is selected from the group consisting of HRAS SEQ ID NOS:63 and 20, NTF3 SEQ ID NOS:2 and 14, MT1A SEQ ID NOS:4 and 16, PAX8 SEQ ID NOS:1 and 13, DIRAS3 SEQ ID NOS:3 and 15, PLAGL1 SEQ ID NOS:7 and 19, SFN SEQ ID NOS:6 and 18, SAT2CHRM1 SEQ ID NOS:9 and 21, MEST SEQ ID NOS:5 and 17, RNR1 SEQ ID NOS:10 and 22, CYP27B1 SEQ ID NOS:11 and 23 and ICAM1 SEQ ID NOS:12 and 24. In certain aspects, the at least one gene sequence is selected from the group consisting of HRAS SEQ ID NOS:63 and 20, NTF3 SEQ ID NOS:2 and 14, MT1A SEQ ID NOS:4 and 16, PAX8 SEQ ID NOS:1 and 13, and PLAGL1 SEQ ID NOS:7 and 19.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows, according to particular exemplary aspects, box plots illustrating associations between semen parameters and level of methylation (PMR) in DNA isolated from 65 study sperm samples. DNA methylation was measured by MethyLight. Methylation targets were sequences specific to the genes HRAS, NTF3, MT1A, PAX8, PLAGL1, DIRAS3, MEST and SFN and the repetitive element Satellite 2 (SAT2CHRM1). P-value for trend over category of semen parameter is given for each plot. Rows: DNA methylation targets; columns: semen parameters.

FIG. 2 shows, according to particular exemplary aspects, cluster analysis of 36 MethyLight targets in 65 study sperm DNA samples. Left: dendrogram defining clusters; rows: 35 methylation targets; columns: 65 study samples ordered left to right on sperm concentration (samples A-G were also included in Illumina analyses (see FIG. 3)) with poor to good concentration (blue), motility (purple), and morphology (green) represented by darkest to lightest hue; body of figure: standardized PMR values represented lowest to highest as yellow to red. X=missing.

FIG. 3 shows, according to particular exemplary aspects, Results of Illumina analysis of 1,421 autosomal sequences in DNA isolated from sperm and buffy coat. Seven study sperm samples (A-G; ordered left to right on sperm concentration), screening sperm (S), two buffy coat (1-2). Level of DNA methylation scored as β-value. Color: β-value for column sample at row sequence (green: β<0.1; yellow: 0.1≦β≦0.25; orange 0.25<β≦30.5; red: β>0.5). MI and PI: maternally and paternally imprinted genes (black bar). Sequences assigned to tertile of median β-value among buffy coat DNA samples (I, II, III) and sorted within tertile on median β-value among sperm DNA samples. Box 1: sequences with sperm-specific DNA methylation; Box 2: sequences with buffy coat-specific DNA methylation.

FIGS. 4 a, b and c schematically depict mammalian primordial germ line development, showing that primordial germ cells undergo extensive epigenetic reprogramming during development, where widespread erasure of DNA methylation occurs in primordial germ cells as they migrate to the gonadal ridges). After the testes form, de novo DNA methylation establishes male-specific DNA methylation during germ cell maturation and spermatogenesis (see FIGS. 4 a and b). According to particular aspects of the present invention, DNA methylation of sperm from men with poor fertility is consistent with disruption of the widespread erasure of DNA methylation that occurs early in the development of germ cells (see FIG. 4 c).

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Overview. There have been several prior art attempts in the art to assess sperm DNA methylation together with either sperm quality or fertility outcomes. However, the measures of DNA methylation used were limited, consisting of either a nonspecific genome-wide measure [18], or small and specialized subsets of DNA methylation targets [19-21].

Specifically, in the only study prior art study addressing the relationship between DNA methylation and fertility outcomes, immunostaining was used to measure genome-wide levels of DNA methylation in samples of ejaculated sperm collected for conventional in vitro fertilization (IVF) [18], and no association was observed between sperm DNA methylation and either fertilization rate or embryo quality in 63 IVF cycles. There was, however, a possible association with pregnancy rate after transfer of good quality embryos. Interpretation of these results is limited by both small sample size and the use of a single summary measure of genome-wide DNA methylation.

Moreover, with respect to the prior art studies [19-21] with small and specialized subsets of DNA methylation targets, sequence-specific measures were used to investigate the relationship between methylation of human sperm DNA and spermatogenesis. One study assessed DNA from spermatogonia and spermatocytes microdissected from seminiferous tubules of biopsied testicular tissue with spermatogenic arrest. DNA profiles consistent with correctly established paternal imprints were reported in all samples [19]. In the remaining two studies [20 and 21], DNA profiles were measured at specific DMRs associated with each of two genes, one paternally and one materially imprinted, and the resulting profiles were related to concentration of ejaculated sperm, an indicator of sperm quality. One of these studies reported correctly erased maternal imprints and correctly established paternal imprints in DNA from sperm of low concentration [21]. By contrast, the second reported that although maternal imprinting of MEST was correctly erased in DNA from sperm of low concentration, methylation at an H19 sequence typically de novo methylated in spermatogenesis was incomplete in these samples [20]. No compelling explanation was offered for the apparently differing results of these studies. It is noteworthy, however, that each addressed sequences of only one or two imprinted genes, an extremely small and specialized subset of DNA methylation targets in the human genome. Data from these published studies could not, therefore, have revealed a disruption involving large numbers of genes, or shown that genes that are not imprinted are also affected.

Particular aspects provide methods for determining or diagnosing abnormal sperm or fertility, comprising: obtaining a sample of human sperm DNA from a test subject; determining, using the genomic DNA of the sample, the methylation status of at least one CpG dinucleotide sequence of at least one gene sequence selected from the group consisting of HRAS, NTF3, MT1A, PAX8, DIRAS3, PLAGL1, SFN, SAT2CHRM1, MEST, RNR1, CYP27B1 and ICAM1; and determining, based on the methylation status of the at least one CpG sequence, the presence or diagnosis of abnormal sperm or fertility with respect to the test subject. In certain embodiments the at least one gene sequence is selected from the group consisting of HRAS SEQ ID NOS:63 and 20, NTF3 SEQ ID NOS:2 and 14, MT1A SEQ ID NOS:4 and 16, PAX8 SEQ ID NOS:1 and 13, DIRAS3 SEQ ID NOS:3 and 15, PLAGL1 SEQ ID NOS:7 and 19, SFN SEQ ID NOS:6 and 18, SAT2CHRM1 SEQ ID NOS:9 and 21, MEST SEQ ID NOS:5 and 17, RNR1 SEQ ID NOS:10 and 22, CYP27B1 SEQ ID NOS:11 and 23 and ICAM1 SEQ ID NOS:12 and 24.

In particular aspects at least on CpG dinucleotide sequence within an amplicon is determined. In preferred aspects, the at least one amplicon sequence is selected from the group consisting of: HRAS SEQ ID NOS:20, NTF3 SEQ ID NO: 14, MT1A SEQ ID NO:16, PAX8 SEQ ID NO:13, DIRAS3 SEQ ID NO:15, PLAGL1 SEQ ID NO:19, SFN SEQ ID NO:18, SAT2CHRM1 SEQ ID NO:21, MEST SEQ ID NO:17, RNR1 SEQ ID NO:22, CYP27B1 SEQ ID NO:23 and ICAM1 SEQ ID NO:24.

Preferably, the amplicon is part of a contiguous CpG island sequence. In preferred aspects, the CpG island sequence sequence is selected from the group consisting of: HRAS SEQ ID NOS:63, NTF3 SEQ ID NO:2, MT1A SEQ ID NO:4, PAX8 SEQ ID NO:1, DIRAS3 SEQ ID NO:3, PLAGL1 SEQ ID NO:7, SFN SEQ ID NO:6, SAT2CHRM1 SEQ ID NO:9, MEST SEQ ID NO:5, RNR1 SEQ ID NO:10, CYP27B1 SEQ ID NO:11 and ICAM1 SEQ ID NO:12.

Coordinate methylation within CpG islands. According to particular aspects, and as recognized in the relevant art, hypermethylation is coordinate within a CpG island. For Example, data (see Eckhardt et al., Nat Genet. 2006 December; 38(12):1378-85. Epub 2006 Oct. 29; incorporated by reference herein in its entirety) has been generated by analyzing methylation (using bisulfite sequencing) in CG-rich regions across entire chromosomes to provide a methylation map of the human genome (at least of the CPG rich regions thereof). To date, these data comprise methylation data of 3 complete human chromosomes (22, 20, and 6) for a variety of different tissues and cell types. Based on these data, for methylation patterns within CpG dense regions, methylation is typically found to be either present for all methylatable cytosines or none. This methylation characteristic or pattern is referred to in the art as “co-methylation” or “coordinate methylation.” The findings of this paper support a “significant correlation” of comethylation over the distance of at least 1,000 nucleotides in each direction from a particular determined CpG within a CpG dense region (see, e.g., page 2, column 2, 1^(st) full paragraph, of Eckhardt et al publication document). Furthermore, such co-methylation forms the basis for long-standing common methods such as MSP and particular MethyLight embodiments that rely on such co-methylation (e.g., as employed herein, the primers and/or probes each typically encompass multiple CpG sequences), and has now been further confirmed over entire chromosomes by Eckhardt et al. Therefore, in view of the teachings of the present specification, there is a reasonable correlation between the claimed coordinately methylated sequences, and the recited methods and exemplary methylation marker sequences.

Measurement of DNA Methylation of the Genomic DNA of Spermatozoa at CpG Islands, DMRs of Imprinted Genes and Repetitive Elements

The present specification describes and discloses the first study ever to investigate the epigenetic state of abnormal human sperm using an extensive panel of DNA methylation assays. Abnormal epigenetic programming of the germ line is herein disclosed as a mechanism compromising fertility of particular men currently diagnosed with idiopathic infertility. Aspects of the present invention indicate that one or more epigenetic processes lead to abnormal spermatogenesis and compromised sperm function.

To assess sperm DNA, methylation at specific targets that are both more numerous and less specialized, a relatively large set of sequence-specific assays was selected for use in the presently disclosed studies and invention.

Specifically, DNA methylation was measured in ejaculated spermatozoa-interrogating sequences in repetitive elements, promoter CpG islands, and differentially methylated regions (DMRs) of imprinted genes. Then, to address the possible role of epigenetic programming in abnormal human spermatogenesis, sequence-specific levels of DNA methylation were related to standard measures of sperm quality.

Applicants' observations indicate a broad epigenetic abnormality of poor quality human sperm in which levels of DNA methylation are elevated at numerous sequences in several genomic contexts. Previous studies of DNA methylation in poor quality sperm interrogated only imprinted loci, measuring methylation of sequences in only one or two genes [19-21].

Aspects of the present invention provide, inter alia, compositions and methods having substantial utility for diagnosing or determining the presence of abnormal sperm or fertility (e.g., comprising at least one of abnormal sperm concentration, abnormal total normal morphology, abnormal motility, abnormal volume, and abnormal viscosity).

As described in the working Example 1, herein below, Applicants initially evaluated 294 MethyLight reactions for the presence of methylation in sperm DNA from an anonymous semen sample obtained from a sperm bank. Standard semen analysis was then conducted on samples collected by 69 men during clinical evaluation of couples with infertility. Thirty seven selected MethyLight reactions were used to assay sperm DNA from 65 of the study samples.

At many of the 37 sequences, methylation levels were elevated in DNA from poor quality sperm. For example, striking associations with each of sperm concentration, motility and morphology were observed for five sequences: HRAS, NTF3, MT1A, PAX8 and the maternally imprinted gene PLAGL1 (FIG. 1). Applicants also found elevated DNA methylation to be significantly associated with poor semen parameters for the DIRAS3 and MEST maternally imprinted genes (FIG. 1).

Associations between results of each of the 37 MethyLight assays and sperm concentration were highly significant for HRAS, NTF3, MT1A, PAX8, DIRAS3 and PLAGL1 and were also significant (somewhat less) for SFN, SAT2CHRM1 and MEST (see Table 1 of Example 1, and see also FIG. 1).

Unsupervised cluster analysis identified three distinct clusters of sequences based on DNA methylation profiles in the 65 samples (FIG. 2). The middle cluster shown in FIG. 2 includes eight of the above nine sequences (all except MT1A) individually associated with semen parameters, and includes not only three sequences that are differentially methylated on imprinted loci, but also three single copy sequences specific to non-imprinted genes, and a repetitive element, Satellite 2 (referred to herein as SAT2CHRM1).

Significantly, this surprising result indicates that sperm abnormalities may be associated with a broad epigenetic defect of elevated DNA methylation at numerous sequences of diverse types, rather than a defect of imprinting alone as previously suggested [20].

To learn more about the possible extent of this apparent defect, the ILLUMINA™ platform was used to conduct DNA methylation analysis of 1,421 sequences in autosomal loci (discussed in more detail under Example 1 herein below). Briefly, the results of the ILLUMINA™ analyses appear in FIG. 3. Box 1 of FIG. 3 identifies 19 sequences with sperm-specific DNA methylation.

Various semen parameters have been correlated herein with abnormal DNA methylation (sperm concentration; total normal morphology; motility, volume, viscosity, etc.). According to preferred aspects, three of these semen parameters show the highest correlations with abnormal DNA methylation: sperm concentration; total normal morphology; and motility. FIG. 2, for example, shows that the corresponding MLL reactions are clustered based on sperm concentration.

Particular aspects of the present invention, therefore, provide marker(s) and marker subsets having utility for determining at least one of (A) abnormal sperm concentration, (B) abnormal morphology, and (C) abnormal motility. With respect to (A), abnormal sperm concentration, markers are provided in the following order of statistical significance from left to right, based on the p-value: HRAS, NTF3, MT1A, PAX8, DIRAS3, PLAGL1, SFN, SAT2CHRM1, MEST, RNR1, and CYP27B1. Nine of these markers have p-values well below 0.05, and therefore are very significant. Additionally provided are the markers, RNR1 and CYP27B1, both having p-values of 0.02, and therefore also provide for utility in this respect.

With respect to (B), abnormal total motile sperm, markers are provided in the following order of statistical significance from left to right, based on the p-value: HRAS, NTF3, MT1A (NTF3 and MT1A equally significant), SAT2CHRM1, DIRAS3, PLAGL1, MEST, PAX8, and SFN. These markers have p-values well below 0.05, and therefore are very significant. Additionally provided are the markers: RNR1 (p-value 0.04) and CYP27B1 and BDNF (both with p-value of 0.05), and therefore also provide for utility in this respect.

With respect to (C), abnormal motility, markers are provided in the following order of statistical significance from left to right, based on the p-value: MT1A, MEST, NTF3, PLAGL1. Additionally provided are the markers PAX8 AND ICAM1 (both having p-values of 0.05), and therefore also provide for utility in this respect.

Improper Erasure of Pre-Existing Methylation

According to particular aspects, only sequence-specific measures of DNA methylation are expected to reveal variation at individual sites, because of the enormous number of methylation targets in the human genome. These include millions of repetitive DNA elements for which methylation is postulated to silence parasitic and transposable activity. There are also large numbers of target sequences corresponding to single copy genes. Examples include thousands of promoter CpG islands for which methylation appears to mediate expression of genes in a tissue- and lineage-specific fashion, and DMRs associated with dozens of imprinted genes for which parent-of-origin DNA methylation marks are believed to mediate monoallelic expression in somatic cells.

As disclosed herein, Applicants' high-throughput analysis addressed hundreds of DNA methylation targets, and was thus designed to reveal methylation defects.

Elevated DNA methylation could, in theory, arise from either de novo methylation or improper erasure of pre-existing methylation. Although Applicants cannot rule out the possibility that processes responsible for de novo methylation are inappropriately activated in abnormal spermatogenesis, according to particular aspects, disruption of erasure is most likely the primary mechanism underlying abnormal spermatogenesis. Widespread erasure of DNA methylation occurs in both the pre-implantation embryo and again, uniquely, in primordial germ cells around the time that they enter the genital ridge. Several factors point to disruption of the second erasure as underlying the defect(s) described herein. Primordial germ cells arise from cells of the proximal epiblast which have themselves embarked upon somatic development, as shown by expression of somatic genes [25,26]. The germ cell lineage must therefore suppress the somatic program, which in mice is accomplished in part by genome-wide erasure of DNA methylation soon after germ cells migrate to the genital ridge [27]. This erasure affects DNA methylation on single copy genes, imprinted genes and repetitive elements [27]. Therefore, disruption of the second, genital ridge erasure most likely results in the type of pattern we observe in poor quality sperm, with elevated levels of DNA methylation at DNA sequences of each of these sequence types. Further, because this second erasure is confined to primordial germ cells, Applicants further reasoned that its disruption would be compatible with normal somatic development.

In humans, primordial germ cells colonize the genital ridge at about 4.5 weeks of gestation. Applicants are not aware of data describing DNA methylation in the human germ line at this date; however, the DMR in MEST at which Applicants found elevated DNA methylation in poor quality sperm is reportedly unmethylated in the male germ line by week 24 of gestation [28]. Potential causes of disrupted erasure have not been investigated. However, weeks 4.5-24 of gestation represent post-implantation stages of development wherein fetal physiology may be influenced by maternal factors and environmental compounds that cross the placenta. Possible origins of male infertility as early as 4.5 weeks of human gestation have not been studied. However, transient in vivo chemical exposure at 7-15 days post conception, which includes the analogous stage of murine development [29,30], results in spermatogenic deficits in rats with grossly normal testes [31] and may be associated with elevated methylation of sperm DNA [32].

Taken together, the observations disclosed herein indicate for the first time that epigenetic mechanisms contribute to a substantial portion of male factor infertility, and provide novel compositions and methods for the diagnosis, detection or determination of abnormal sperm or fertility. Also provided are methods for screening for agents that cause spermatogenic deficits, adbnormal sperm or fertility comprising: obtaining human ES-cell derived primordial germ cells; contacting the germ cells with at least one test agent; culturing the contacted germ cells; obtaining a sample of genomic DNA from the contacted cultured germ cells; determining, using the genomic DNA of the sample, the methylation status of at least one CpG dinucleotide sequence of at least one gene sequence selected from the group consisting of HRAS, NTF3, MT1A, PAX8, DIRAS3, PLAGL1, SFN, SAT2CHRM1, MEST, RNR1, CYP27B1 and ICAM1; and identifying, based on the methylation status of the at least one CpG sequence, at least one test agent that causes spermatogenic deficits, adbnormal sperm or fertility.

Example 1 Sequence-Specific Levels of DNA Methylation were Related to Standard Measures of Sperm Quality Overview

This is the first study ever to describe the epigenetic state of abnormal human sperm using an extensive panel of DNA methylation assays. To assess sperm DNA methylation at specific targets that are both more numerous and less specialized, a relatively larger set of sequence-specific assays was selected for use in the present study. DNA methylation was measured in ejaculated spermatozoa—interrogating sequences in repetitive elements, promoter CpG islands, and differentially methylated regions (DMRs) of imprinted genes. Then, to address the possible role of epigenetic programming in abnormal human spermatogenesis, sequence-specific levels of DNA methylation were related to standard measures of sperm quality.

Materials and Methods

Semen samples. Study semen samples were collected by 69 consecutive men ages 22-49 years who were partners of women undergoing evaluation for infertility at the Endocrine/Infertility Clinic of the Los Angeles County/University of Southern California Keck School of Medicine Medical Center. One additional semen sample was obtained from a sperm bank. The study was approved by the Institutional Review Board of the University of Southern California. Informed consent was not required because this research involved stored materials that had previously been collected solely for non-research purposes and were anonymous to the researchers/authors.

Semen Analysis. Standard semen analysis was performed using WHO criteria and Strict Morphology as previously described [33,34]. Semen volume, sperm concentration and motility, and leukocyte count were measured using the MicroCell chamber (Conception Technologies, San Diego, Calif.). Sperm morphology was assessed with the use of prestained slides (TestSimplets, Spectrum Technologies, Healdsburgh, Calif.), and percentage of morphologically normal sperm was documented. The samples were categorized according to concentration (<5, 5-20, >20 million sperm/ml), motility (<10, 10-50, >50 total motile sperm count (×10⁶)), and morphology (<5%, 5-14%, >14% normal) of sperm [33,35]. Presence of any white blood cells, round cells, or epithelial cells was recorded. Following semen analysis, samples were stored at −30° C. until processing for molecular analysis.

Sperm Separation from Seminal Plasma. Semen samples were allowed to thaw at 37° C. Sperm were separated from seminal plasma using ISOLATE® Sperm Separation Medium (Irvine Scientific, Santa Ana, Calif.), a density gradient centrifugation column designed to separate cellular contaminants (including leukocytes, round cells, and miscellaneous debris) from spermatozoa [24]. Separation was performed according to the manufacturer's protocol [36], and the purity of separated sperm from contaminating cells was documented by light microscopy.

DNA isolation. DNA was isolated from purified sperm as previously described [37], with 0.1×SSC added to the Lysis buffer, and samples incubated at 55° C. over night or longer to complete the lysis procedure.

Laboratory Analysis of DNA Methylation. Sodium bisulfite conversion was performed as previously described [23]. The amount of DNA in each aliquot was normalized, and a bisulfite-dependent, DNA methylation-independent control reaction was performed to confirm relative amounts of DNA in each sample. METHYLIGHT™ analyses were performed as previously described [23]. Reaction IDs and sequences of the primers and probes used in the 294 METHYLIGHT™ reactions are as previously published (see Table 51 (Sections A-B): doi:10.1371/journal.pone.0001289.s001 (0.10 MB PDF; incorporated by reference herein in its entirety). Additionally, according to particular aspects of the present invention, names of preferred markers and respective primers, probes and genomic sequences corresponding to the respective amplicons are listed below in TABLE 1.

TABLE 1 Primers and Probes for exemplary preferred MethyLight Assays. Genomic sequence Forward Reverse Probe Oligo corresponding to Primer Primer Sequence amplicon Sequence Gene (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) HRAS GAGCGATGACG CGTCCACAAAA 6FAM- CGTCCACAAAATGGTTCTGG GAATATAAGTT TAATTCTAAAT CACTCTTACCC ATCAGCTGGATGGTCAGCGC GG CAACTAA ACACCGCCGAC ACTCTTGCCCACACCGCCGG (SEQ ID NO: 46) (SEQ ID NO: 47) G-BHQ-1 CGCCCACCACCACCAGCTTA (SEQ ID NO: 48) TATTCCGTCATCGCTC (SEQ ID NO: 20) NTF3 TTTCGTTTTTG CCGTTTCCGCC 6FAM- CCCCGCCCTTGTATCTCATG TATTTTATGGA GTAATATTC TCGCCACCACG GAGGATTACGTGGGCAGCCC GGATT (SEQ ID NO: 29) AAACTACCCAC CGTGGTGGCGAACAGAACAT (SEQ ID NO: 28) G-BHQ-1 CACGGCGGAAACGG (SEQ ID NO: 30) (SEQ ID NO: 14) MT1A CGTGTTTTCGT CTCGCTATCGC 6FAM- CGTGTTCCCGTGTTACTGTG GTTATTGTGTA CTTACCTATCC TCCACACCTAA TACGGAGTAGTGGGTCCGAG CG (SEQ ID NO: 35) ATCCCTCGAAC GGACCTAGGTGTGGACAGGG (SEQ ID NO: 34) CCACT-BHQ-1 ACAGGCAAGGCGACAGCGAG (SEQ ID NO: 36) (SEQ ID NO: 16) PAX8 CGGGATTTTTT ACCTTTCCCCA 6 FAM- CGGGACCTCCCTGTCGTACC TGTCGTATTTG TACTACCTCCG ACGAACAATTC TGAGAGGAGGGCCTGGCCCG A (SEQ ID NO: 26) ACGAACCAAAC TGAACTGCCCGTACACGGAG (SEQ ID NO: 25) CCTCCT-BHQ-1 GCAGCATGGGGAAAGGC (SEQ ID NO: 27) (SEQ ID NO: 13) DIRAS3 GCGTAAGCGGA CCGCGATTTTA 6 FAM- GCGCAAGCGGAATCTATGCC ATTTATGTTTG TATTCCGACTT CGCACAAAAAC TGTTACCCACACTCCCTGCG T (SEQ ID NO: 32) GAAATACGAAA CCCCCGCACCCCGCTCCTGT (SEQ ID NO: 31) ACGCAAA- GCGCAAGTCGGAATATAAAA BHQ-1 CCGCGG (SEQ ID NO: 33) (SEQ ID NO: 15) PLAGL1 ATCGACGGGTT CTCGACGCAAC 6FAM- ACCGACGGGCTGAATGACAA GAATGATAAAT CATCCTCTT ACTACCGCGAA ATGGCAGATGCCGTGGGCTT G (SEQ ID NO: 44) CGACAAAACCC TGCCGCCCGCGGCAGCCAAG (SEQ ID NO: 43) ACG-BHQ-1 AGGATGGCTGCGCCGAG (SEQ ID NO: 45) (SEQ ID NO: 19) SFN GAGGAGGGTTC ATCGCACACGC 6FAM- GAGGAGGGCTCGGAGGAGAA GGAGGAGAA CCTAAAACT TCTCCCGATAC GGGGCCCGAGGTGCGTGAGT (SEQ ID NO: 40) (SEQ ID NO: 41) TCACGCACCTC ACCGGGAGAAGGTGGAGACT GAA-BHQ-1 GAGCTCCAGGGCGTGTGCGA (SEQ ID NO: 42) C (SEQ ID NO: 18) SAT2CHRM1 TCGAATGGAAT CCATTCGAATC 6FAM- TCGAATGGAATCAACATCCA TAATATTTAAC CATTCGATAAT CGATTCCATTC ACGGAAAAAAACGGAATTAT GGAAAA TCT GATAATTCCGT CGAATGGAATCGAAGAGAAT (SEQ ID NO: 49) (SEQ ID NO: 50) TT-MGBNFQ CATCGAATGGACCCGAATGG (SEQ ID NO: 51) (SEQ ID NO: 21) MEST CGGCGTTCGGT CACACTCACCT 6FAM- CGGCGCCCGGTGCTCTGCAA GTTTTGTAA ACGAAAACGAT ACGCACCATAA CGCTGCGGCGGGCGGCATGG (SEQ ID NO: 37) CTC CATACC-BHQ-1 GATAACGCGGCCATGGTGCG (SEQ ID NO: 38) (SEQ ID NO: 39) CCGAGATCGCCTCCGCAGGT GAGTGTG (SEQ ID NO: 17) RNR1 CGTTTTGGAGA AAACAACGCCG 6FAM- CGCTCTGGAGACACGGGCCG TACGGGTCG AACCGAA ACCGCCCGTAC GCCCCCTGCGTGTGGCACGG (SEQ ID NO: 52) (SEQ ID NO: 53) CACACGCAAA- GCGGCCGGGAGGGCGTCCCC BHQ-1 GGCCCGGCGCTGCTC (SEQ ID NO: 54) (SEQ ID NO: 22) CYP27B1 GGGATAGTTAG CCGAATATAAC 6FAM- GGGACAGCCAGAGAGAACGG AGAGAACGGAT CACACCGCC CCAACCTCAAC ATGCCCATGAAATAAGGAAA GTTT (SEQ ID NO: 56) TCGCCTTTTCC AGGCGAGTTGAGGCTGGGGG (SEQ ID NO: 55) BHQ-1 CGGTGTGGCTACACTCGG (SEQ ID NO: 57) (SEQ ID NO: 23) ICAM1 GGTTAGCGAGG TCCCCTCCGAA 6FAM- GGCCAGCGAGGGAGGATGAC GAGGATGATT ACAAATACTAC TTCCGAACTAA CCTCTCGGCCCGGGCACCCT (SEQ ID NO: 58) AA CAAAATACCCG GTCAGTCCGGAAATAACTGC (SEQ ID NO: 59) BHQ-1 AGCATTTGTTCCGGAGGGGA (SEQ ID NO: 60) (SEQ ID NO: 24)

Thirty-five METHYLIGHT™ reactions were selected for analysis of study sperm DNA samples based on cycle threshold (C(t)) values from analysis of the anonymous sample of sperm DNA. In brief, C(t) value is the PCR cycle number at which the emitted fluorescence is detectable above background levels. The C(t) value is inversely proportional to the amount of each methylated locus in the PCR reaction well, such that a low C(t) value suggests that the interrogated sequence is highly methylated. C(t) values of 35 or less were interpreted as an indication that a given sequence was methylated in the anonymous sample and selected 33 reactions on this basis. Three additional reactions were included, for which C(t) values slightly exceeded 35. Two (CYP27B1 and HOXA10) were selected based on gene function potentially related to fertility, and one (a non-CpG island reaction for IFNG) based on prior observation by applicants of hypomethylation in tumor versus normal tissue. When multiple reactions for a single locus resulted in C(t) values of less than 35, we selected only the reaction with the lowest C(t) value. Results of METHYLIGHT™ analysis were scored as PMR values as previously defined [23]. Following METHYLIGHT™ analyses, DNA remained from a subset of abnormal samples with greater sperm concentration. ILLUMINA™ analysis was performed on sodium bisulfite-converted sperm DNA of selected remaining samples, the anonymous semen sample, and purchased buffy coat DNA (HemaCare® Corporation, Van Nuys, Calif.) at the USC Genomics Core. Sodium bisulfite conversion for ILLUMINA™ assay was performed using the EZ-96 DNA Methylation Kit™ (ZYMO Research) according to manufacturer's protocol. Illumina Methods and reagents are as previously described [38]. The primer names and probe IDs are listed as previously published (see Table S2; doi:10.1371/journal.pone.0001289.s002 (0.20 MB PDF; incorporated by reference herein in its entirety), identifying 1,421 autosomal sequences of the GoldenGate Methylation Cancer Panel I, more fully described elsewhere [39,40]. Results of ILLUMINA™ assays were scored as β-values [38]. Relevant amplicons and CpG islands are provided below in TABLE 2 below.

Statistical association analyses of METHYLIGHT™ data. Associations between the ranked METHYLIGHT™ data and categorized semen values (Table 1) were tested using simple linear regression, with the semen characteristic categories scored as 0: low, 1: mid, 2: high. For selected sequences, boxplots of the methylation values (on the log(PMR+1) scale) are shown in FIG. 1. The top and bottom of the box denote the 75^(th) and 25^(th) percentiles, and the white bar the median. Whiskers are drawn to the observation farthest from the box that lies within 1.5 times the distance from the top to the bottom of the box, with values falling outside the whiskers denoted as lines. Results of this analysis were included in FIG. 1 for sequences associated with sperm concentration using the Benjamini and Hochberg procedure [41] to control the false discovery rate at 5%.

Statistical cluster analysis of METHYLIGHT™ data. Hierarchical cluster analysis of 36 loci was performed, using correlation to measure the distance between any two loci and Ward's method of linkage [42]. SASH1 was omitted from the cluster analysis because only a single sample showed positive methylation. The 65 study samples were ordered from left to right by increasing semen concentration.

TABLE 2 Exemplary, preferred amplicons and CpG islands HUGO Gene Reaction Nomen- Previously Source of UniGene Reaction Alternate Gene Number clature Published? published reaction Number ID Name HB-144 HRAS Yes Widschwendter, M. Hs.37003 H-HRAS- V-Ha-ras Harvey rat et al Cancer Res M1B sarcoma viral 64, 3807-3813 oncogene homolog (2004) (HRAS); HRAS1 HB-251 NTF3 Yes Weisenberger, D. J. Hs.99171 H-NTF3- Neurotrophin 3 et al Nature Genet M1B 38, 787-793 (2006). HB-205 MT1A Yes Weisenberger, D. J. Hs.655199 H-MT1A- Metallothionein et al Nature Genet M1B 1A/Metallothionein-I 38, 787-793 (2006). HB-212 PAX8 No Hs.469728 H-PAX8- Paired Box Gene 8/ M3B PAX8, Paired Domain Gene 8, PPARG Fusion Gene HB-043 DIRAS3 Yes Fiegl, H. et al Hs.194695 H-DIRAS3- Ras homolog gene Cancer Epidemiol M1B family, member BioMark Prev I/NOEY2; DIRAS 13, 882-888 (2004) family, GTP-binding RAS-like 3 (ARHI) HB-199 PLAGL1 Yes Weisenberger, D. J. Hs.444975 H- Pleiomorphic et al Nature Genet PLAGL1- adenoma gene-like 38, 787-793 M1B 1/LOT1/Zac1 (2006). HB-174 SFN Yes Weisenberger, D. J. Hs.523718 H-SFN- Stratifin/14-3-3 et al Nature Genet M1B protein sigma 38, 787-793 (2006). HB-289 SAT2CHRM1 Yes Weisenberger, D. J. N/A H- SATELLITE 2 et al Nucleic Acids SAT2CHRM1- CHROMOSOME 1 Res 33, 6823-6836 M1M (2005) HB-493 MEST No Hs.270978 H-MEST- PEG1 M2B HB-071 RNR1 Yes Muller, H. M. et al. N/A H-RNR1- Ribosomal RNA Cancer Lett209, M1B 231-236 (2004) HB-076 ICAM1 Yes Ehrlich, M. et al. Hs.643447 H- Intercellular Oncogene 21, ICAM1B- adhesion molecule 1 6694-6702 (2002) M1B (CD54), human rhinovirus receptor HB-223 CYP27B1 Yes Weisenberger, D. J. Hs.524528 H- cytochrome P450, et al Nature Genet CYB27B1- family 27, subfamily 38, 787-793 M1B B, polypeptide 1 (2006). HUGO GenBank Length Transcription Gene Accession mRNA of Start Reaction Nomen- Chromosomal Number; accession Parallel/ Sequence (GenBank Number clature Location version number Antiparallel (bp) Numbering) HB-144 HRAS 11p15.5 AC137894; NM_176795; Antiparallel 165000 157238 AC137894.5 NM_176795.3 GI: 29650323 GI: 194363760 HB-251 NTF3 12p13 AC135585; NM_002527; Parallel 35700 7048 AC135585.2 NM_002527.4 GI: 24371348 GI: 156630993 HB-205 MT1A 16q13 AC106779; NM_005946; Parallel 158297 18787 AC106779.3 NM_005946.2 GI: 18483433 GI: 71274112 HB-212 PAX8 2q12 AC016683; S77905; Antiparallel 179937 116171 AC016683.7 S77905.1 GI: 11136842 GI: 998702 HB-043 DIRAS3 1p31 AF202543; U96750; Parallel 7242 2053 AF202543.1 U96750.1 GI: 11493727 GI: 4100354 (NG_011753.1 (NM_004675.2 GI: 226053757) GI: 58530880) HB-199 PLAGL1 6q24-q25 AL109755; U72621; Antiparallel 89669 53085 AL109755.14 U72621.4 GI: 6165524 GI: 21930302 (NG_009384.1 (Variants 1-8) GI: 221136778( HB-174 SFN 1p35.3 AF029081; BC023552; Parallel 10034 8563 AF029081.1 BC023552.2 GI: 2702352 GI: 33873404 HB-289 SAT2CHRM1 1 X72623; N/A; Parallel 1352 N/A X72623.1 GI: 599824 HB-493 MEST 7q32.2 NC_000007; NM_177524; Parallel 20084 5893 NC_000007.13 NM_177524.1 GI: 224589819 GI: 29294634 HB-071 RNR1 13p12 X01547; N/A Parallel 850 482 X01547.1 GI: 35916 HB-076 ICAM1 19p13.3-p13.2 AC011511; BC015969; Parallel 156503 85732 AC011511.12 BC015969.2 GI: 21747446 GI: 33869582 HB-223 CYP27B1 12q14.1 AY288916; AB005038; Parallel 7587 1324 AY288916.1 AB005038.1 GI: 30527185 GI: 2626736 Amplicon Amplicon Start End Mean Location Location Distance Relative to Relative to from Amplicon Amplicon Transcription Transcription Transcription HUGO Location Location Start Start Start Gene Start End (bp, (bp, (bp, Reaction Nomen- (GenBank (GenBank GenBank GenBank GenBank Number clature Numbering) Numbering) sequence) Sequence) sequence) HB-144 HRAS 156015 155920 1223 1318 1271 HB-251 NTF3 7503 7576 455 528 492 HB-205 MT1A 18175 18254 −612 −533 −573 HB-212 PAX8 72708 72632 43463 43539 43501 HB-043 DIRAS3 1953 2038 −100 −15 −58 HB-199 PLAGL1 53045 52969 40 116 78 HB-174 SFN 8848 8928 285 365 325 HB-289 SAT2CHRM1 1074 1153 N/A N/A N/A HB-493 MEST 6057 6144 164 251 207 HB-071 RNR1 219 293 −263 −189 −226 HB-076 ICAM1 85597 85676 −135 −56 −96 HB-223 CYP27B1 1728 1805 404 481 443 HUGO Amplicon Amplicon Location of Gene Location Start Location End UCSC UCSC Amplicon in Gene Reaction Nomen- (UCSC (UCSC Strand Assembly (e.g., promoter, Number clature Numbering) Numbering) (+/−) Date exon) HB-144 HRAS 524232 524327 + May 2004 Exon2 HB-251 NTF3 5473982 5474055 + May 2004 Exon1 HB-205 MT1A 55229471 55229550 + May 2004 Promoter HB-212 PAX8 113709183 113709259 + May 2004 Exon 9 HB-043 DIRAS3 68228349 68228434 − May 2004 Promoter (in Exon3) HB-199 PLAGL1 1443711135 144371211 + May 2004 Exon1 HB-174 SFN 26874056 26874136 + May 2004 Exon1 HB-289 SAT2CHRM1 no perfect no perfect match May 2004 N/A match HB-493 MEST 129919339 129919425 + March 2006 exon1/intron1 HB-071 RNR1 N/A N/A May 2004 Promoter HB-076 ICAM1 10242630 10242709 + May 2004 Promoter HB-223 CYP27B1 56446731 56446808 − May 2004 Exon1 500 (approx. ± 250) bp sequence Location of Location of comprising Estimated CpG Island CpG Island CpG Island HUGO Gene amplicon CpG Length (GenBank) Start End Reaction Nomen- (Genbank Island (SEQ ID NO:) (GenBank (GenBank Number clature sequence) yes/no (>0.6 CpG:GpC) numbering) numbering) HB-144 HRAS 155726-156225 (Yes) 3354 (SEQ ID NO: 63) 156171 159524 HB-251 NTF3 7301-7800 Yes  609 (SEQ ID NO: 2) 7246 7854 HB-205 MT1A 18201-18700 Yes 1209 (SEQ ID NO: 4) 17842 19050 HB-212 PAX8 72426-72925 Yes 1250 (SEQ ID NO: 1) 73859 72610 HB-043 DIRAS3 1751-2250 Yes  552 (SEQ ID NO: 3) 1804 2355 HB-199 PLAGL1 52751-53250 Yes 1478 (SEQ ID NO: 7) 53667 52190 HB-174 SFN 8637-9136 Yes  661 (SEQ ID NO: 6) 8684 9344 HB-289 SAT2CHRM1  851-1350 (Yes)  (500 (SEQ ID NO: 9)) N/A N/A HB-493 MEST Yes 2799 (SEQ ID NO: 5) 4293 7091 HB-071 RNR1  1-500 yes  850 (SEQ ID NO: 10) 1 850 HB-076 ICAM1 85376-85875 Yes 2038 (SEQ ID NO: 12) 84047 86084 HB-223 CYP27B1 1501-2000 yes  747 (SEQ ID NO: 11) 1345 2091 HUGO Amplicon Bisulfite Gene Start Conversion: Reaction Nomen- relative to Top/Bottom Number clature CGI start Reaction Type Strand HB-144 HRAS N/A Methylated Bottom HB-251 NTF3 257 Methylated Top HB-205 MT1A 333 Methylated Top HB-212 PAX8 1151 Methylated Top HB-043 DIRAS3 149 Methylated Top HB-199 PLAGL1 622 Methylated Top HB-174 SFN 116 Methylated Top HB-289 SAT2CHRM1 N/A Methylated Top HB-493 MEST 1764 Methylated Top HB-071 RNR1 219 Methylated Top HB-076 ICAM1 1685 Methylated Top HB-223 CYP27B1 383 Methylated Top

Display of ILLUMINA™ data. ILLUMINA™ data were displayed graphically in FIG. 3 with results for study samples ordered left to right in columns by sperm concentration. Rows corresponding to each of the 1,421 sequences were divided into three tertiles of median β-value among buffy coat DNA samples (I, II, III), then sorted within tertile by median β-value among all sperm DNA samples. Box 1 contains all sequences tertile I with median β-value among sperm DNA samples >0.5; box 2 contains all sequences within tertile III with median β-value among sperm DNA samples <0.1. Maternal or paternal imprinting status of each locus was scored according to the categorization of R. Jirtle [43]. All sequences specific to genes imprinted in humans were individually reviewed to determine whether they have been reported as belonging to a DMR for which parent of origin marks are maintained by DNA methylation [44-66]. Sequences meeting these criteria were scored as maternally imprinted (MI) or paternally imprinted (PI) with an indicator set for each on FIG. 3.

Results

Standard semen analysis was conducted on samples collected by 69 men during clinical evaluation of couples with infertility. Among the 69 samples, semen volume ranged from 0.5 to 7.8 ml; total count 0 to 864 million sperm; total motile count 0 to 396.3 million sperm; and percentage normal sperm forms 0 to 26%. Four samples were found to be azoospermic and excluded from subsequent analysis of DNA methylation.

Applicants evaluated 294 METHYLIGHT™ reactions for the presence of methylation in sperm DNA from an anonymous semen sample obtained from a sperm bank. Primers and probes were as previously published (see Table 51 (Sections A-B), found at doi:10.1371/journal.pone.0001289.s001 (0.10 MB PDF); incorporated by reference herein in its entirety; Primers, probes and reaction IDs for 294 MethyLight Assays: Group A, used in screening procedure and analysis of 65 study samples; Group B, used only in screening procedure; and Group C, new assays designed to DMRs of maternally imprinted genes and used only in analysis of 65 study samples.

The 35 selected reactions of Table 51A were used to assay sperm DNA from 65 study samples.

At many of the 35 sequences methylation levels were elevated in DNA from poor quality sperm. For example, striking associations with each of sperm concentration, motility and morphology were observed for five sequences: HRAS, NTF3, MT1A, PAX8 and PLAGL1 (FIG. 1).

PLAGL1 is maternally imprinted. Our METHYLIGHT™ assay for this gene interrogates a differentially methylated CpG island [22]. To determine whether other maternally imprinted genes are methylated in abnormal sperm, METHYLIGHT™ was used to interrogate the differentially methylated sequence of DIRAS3. At this sequence greater DNA methylation was also observed in samples with poorer semen parameters (FIG. 1, row 6). These results appeared to conflict with those of Marques et al [20] who reported no association between low sperm count and methylation of a DMR in a third maternally imprinted gene, MEST. We therefore used METHYLIGHT™ to assess the methylation status of a differentially methylated MEST sequence investigated by these authors [20], and found elevated DNA methylation to be significantly associated with poor semen parameters (FIG. 1), in agreement with our PLAGL1 and DIRAS3 results.

After correction for multiple comparisons, estimated associations between results of each of the 37 METHYLIGHT™ assays and sperm concentration were highly significant for HRAS, NTF3, MT1A, PAX8, DIRAS3 and PLAGL1 and marginally significant for SFN, SAT2CHRM1 and MEST (Table 3, FIG. 1).

TABLE 3 Trend p-values for associations between MethyLight results and semen parameters (see Methods). Parameter of Standard Semen Analysis MethyLight Reaction Concentration Motility Morphology *HRAS.HB.144 0.00006 0.00001 0.06265 *NTF3.HB.251 0.00029 0.00026 0.00464 MT1A.HB.205 0.00048 0.00026 0.00119 *PAX.8.HB.212 0.00086 0.00405 0.05143 *DIRAS3.HB.043 0.00109 0.00159 0.06016 *PLAGL1.HB.199 0.00213 0.00255 0.01951 *SFN.HB.174 0.00307 0.00804 0.79899 *SAT2CHRM1.HB.289 0.00448 0.00109 0.06793 *MEST.HB.493 0.00711 0.00373 0.00359 RNR1.HB.071 0.02 0.04 0.89 CYP27B1 0.02 0.05 0.10 MADH3.HB.053 0.09 0.15 0.35 BDNF.HB.257 0.11 0.05 0.26 PSEN1.HB.263 0.16 0.27 0.81 CGA.HB.237 0.23 0.34 0.93 SERPINB5.HB.208 0.23 0.64 0.80 ICAM1.HB.076 0.24 0.29 0.05 MINT1.HB.161 0.24 0.60 0.34 PTPN6.HB.273 0.24 0.09 0.08 ALU.HB.296 0.25 0.29 0.87 CYP1B1.HB.239 0.28 0.42 0.61 SP23.HB.301 0.28 0.48 0.48 IFNG.HB.311 0.33 0.22 0.93 C9.HB.403 0.37 0.35 0.89 GP2.HB.400 0.41 0.39 0.94 GATA4.HB.325 0.45 0.20 0.12 UIR.HB.189 0.48 0.47 0.70 TFF1.HB.244 0.48 0.96 0.93 LDLR.HB.219 0.51 0.39 0.11 SASH1.HB.085 0.51 0.15 0.15 ABCB1.HB.051 0.54 0.27 0.16 HOXA10.HB.270 0.63 0.84 0.13 MTHFR.HB.058 0.70 0.38 0.43 LINE1.HB.330 0.87 0.47 0.14 LZTS1.HB.200 0.90 0.95 0.73 SMUG1.HB.086 0.90 0.36 0.76 ^(‡)IGF2.HB.345 0.91 0.71 0.11 *Belongs to cluster 2 (see FIG. 2). ^(‡)Assay interrogates a non-differentially methylated sequence. Trends were assessed over the following categories of semen parameters: Concentration (<5, 5-20, >20 × 10⁶ sperm per ml), Morphology (<5%, 5-14%, >14% normal sperm forms), Motility (<10, 10-50, >50 total motile sperm count (×10⁶)).

Applicants then subjected METHYLIGHT™ data from 36 of the assays to unsupervised cluster analysis. (Data for SASH1 were not included, because methylation at this sequence was detected in only one sample.) This analysis identified three distinct clusters of sequences based on DNA methylation profiles in the 65 samples (FIG. 2). Notably, the middle cluster shown in FIG. 2 includes eight of the nine sequences (all except MT1A) individually associated with semen parameters. This middle cluster includes not only three sequences that are differentially methylated on imprinted loci, but also three single copy sequences specific to non-imprinted genes, and a repetitive element, Satellite 2 [23] (reaction named SAT2CHRM1).

Significantly, this surprising result indicates that sperm abnormalities may be associated with a broad epigenetic defect of elevated DNA methylation at numerous sequences of diverse types, rather than a defect of imprinting alone as previously suggested [20].

To learn more about the possible extent of this apparent defect, the ILLUMINA™ platform was used to conduct DNA methylation analysis of 1,421 sequences in autosomal loci. Included in this analysis was: DNA from the anonymous sperm sample used in the METHYLIGHT™ screen (FIG. 3, columns S); two purchased samples of buffy coat DNA allowing for observation of methylation patterns in somatic cells (FIG. 3, columns 1-2), and seven study sperm DNA samples remaining after METHYLIGHT™ analysis (FIGS. 2-3, columns A-G).

Results of ILLUMINA™ analyses appear in FIG. 3. A large number of genes were similarly methylated in both sperm DNA and buffy coat DNA (blue regions on the left bar, I; red regions on the right bar, III), while others tended to be more methylated in DNA isolated from only one of these cell types. Boxes enclose sequences for which we observed particularly strong patterns of cell type-specific methylation. Box 1 identifies 19 sequences with sperm-specific DNA methylation. At these sequences, methylation profiles of all DNA from samples of study sperm (A-G) closely resemble those from the anonymous sperm sample and differ greatly from those of buffy coat DNA. Box 2 identifies 102 sequences with buffy coat-specific DNA methylation. This set is larger in number than the sperm-specific set, as expected, given that sperm DNA is reportedly hypomethylated compared with somatic cell DNA [14]. The buffy coat-specific set comprises 7.2% of the 1,421 sequences including the majority of DMRs associated with imprinted genes that are on the Illumina panel. At many buffy coat-specific sequences, DNA methylation was elevated in study sperm DNA, most notably in sample A that had been isolated from sperm with the lowest concentration among samples A-G. Methylation of sample A DNA is elevated (β>0.1) at 76 of the 102 sequences in box 2, including all 10 that are known DMRs associated with imprinted genes.

Several factors assure us that our observations did not arise from somatic cell contamination of separated sperm samples [21]. Somatic cells are far larger than sperm and readily identified by microscopic evaluation of semen samples. Even if somatic cells are present in the neat ejaculate, the ISOLATE® sperm separation technique is specifically designed to separate spermatozoa from somatic cells and miscellaneous debris [24]. Moreover, although microscopic evaluation of semen samples conducted before sperm separation identified white blood cells in five of the 65 neat semen samples, excluding results on these five samples from statistical analyses had minimal effect on associations between DNA methylation and semen parameters, and DNA from these samples were excluded from ILLUMINA™ assays.

Various semen parameters have been correlated with abnormal DNA methylation (sperm concentration; total normal morphology; motility, volume, viscosity, etc.). According to preferred aspects, three of these semen parameters show the highest correlations with abnormal DNA methylation: sperm concentration; total normal morphology; and motility. FIG. 2, for example, shows that the corresponding MLL reactions are clustered based on sperm concentration.

Particular preferred aspects, therefore, provide marker(s) and marker subsets having utility for determining at least one of abnormal sperm concentration, abnormal morphology, and abnormal motility.

In particular aspects, with respect to (A) abnormal sperm concentration, markers are provided in the following order of statistical significance from left to right, based on the p-value: HRAS, NTF3, MT1A, PAX8, DIRAS3, PLAGL1, SFN, SAT2CHRM1, and MEST. All of these nine markers have p-values well below 0.05, and therefore, all nine are very significant. Additionally provided are two more markers, RNR1 and CYP27B1, both have p-value of 0.02, that are therefore also of utility in this respect.

In particular aspects, with respect to (B) abnormal total motile sperm, markers are provided in the following order of statistical significance from left to right, based on the p-value: HRAS, NTF3, MT1A (NTF3 and MT1A equally significant), SAT2CHRM1, DIRAS3, PLAGL1, MEST, PAX8, & SFN. Again, these have very significant p-values. Additionally provided are three more markers: RNR1(p-value 0.04) and CYP27B1, BDNF, both with p-value of 0.05, that are therefore also of utility in this respect.

In particular aspects, with respect to (C) abnormal motility, markers are provided in the following order of statistical significance from left to right, based on the p-value: MT1A, MEST, NTF3, PLAGL1. Additionally, PAX8 AND ICAM1 both have p-values of 0.05, and are thus also of utility in this respect.

Example 2 Additional Aspects Provide Methods for Screening for Agents that Cause Spermatogenic Deficits, Abnormal Sperm or Abnormal Fertility Overview

As stated herein above, this is the first study ever to describe the epigenetic state of abnormal human sperm using an extensive panel of DNA methylation assays. According to additional aspects, Applicants data has provided novel methylation-based markers for abnormal human sperm and/or fertility.

As recognized in the art, transient in vivo chemical exposure at 7-15 days post conception, which includes the analogous stage of murine development [29,30], results in spermatogenic deficits in rats with grossly normal testes [31] but likely associated with elevated methylation of sperm DNA [32].

According to additional aspects, therefore, Applicants' data provides for methods for screening for agents that cause spermatogenic deficits, abnormal sperm or abnormal fertility. In particular aspects, ES-cell derived primordial germ cells are exposed to chemical test agents, followed by CpG methylation analysis as described and provided for herein, to allow for a high-throughput screening assay to test and identify agents that cause spermatogenic deficits, abnormal sperm or abnormal fertility. Culturing of embryonic stem (ES) cells to efficiently provide for primordial germ cells is known in the art. For example, human embryonic stem (ES) cells are propagated on mouse embryo fibroblast feeder cells as described (67). A multistep induction procedure incorporating several previously described protocols can be used to convert ES cells into primordial germ cells at high efficiency. For example, ES cells are treated with bone morphogenetic protein-2 for a brief 24 period in combination with activin and FGF-2 in chemically defined medium. After 24 hours the BMP-2 is removed and retinoic acid is added. As will be appreciated in the art, a range of doses of each factor may be employed in a matrix design over a variable time course to optimize the yield of c-kit positive/placental alkaline phosphatase positive cells. These cells are isolated by flow cytometry and subjected to Q-RTPCR to analyze for the presence of primordial germ cell and gonocyte specific genes such as VASA. According to particular aspects, up to 10% of the treated cells are vasa positive following optimal treatment. Primordial germ cells and gonocytes may also be isolated from embryonic and fetal gonads by the use of c-kit and placental alkaline phosphatase in combination with flow cytometry, following collagenase and Tryple Express™ digestion of the tissue.

Particular aspects, therefore, provide methods for screening for agents that cause spermatogenic deficits, abnormal sperm or abnormal fertility comprising: obtaining human ES-cell derived primordial germ cells; contacting the germ cells or descendants thereof, with at least one test agent; culturing the contacted germ cells or the descendants thereof under conditions suitable for germ cell proliferation or development; obtaining a sample of genomic DNA from the contacted cultured germ cells or the descendants thereof; determining, using the genomic DNA of the sample, the methylation status of at least one CpG dinucleotide sequence of at least one gene sequence selected from the group consisting of HRAS, NTF3, MT1A, PAX8, DIRAS3, PLAGL1, SFN, SAT2CHRM1, MEST, RNR1, CYP27B1 and ICAM1; and identifying, based on the methylation status of the at least one CpG sequence, at least one test agent that causes at least one of spermatogenic deficits, abnormal sperm, and abnormal fertility. In certain embodiments, the determined methylation status of the at least one CpG sequence is hypermethylation. In preferred embodiments, the at least one gene sequence is selected from the group consisting of HRAS SEQ ID NOS:63 and 20, NTF3 SEQ ID NOS:2 and 14, MT1A SEQ ID NOS:4 and 16, PAX8 SEQ ID NOS:1 and 13, DIRAS3 SEQ ID NOS:3 and 15, PLAGL1 SEQ ID NOS:7 and 19, SFN SEQ ID NOS:6 and 18, SAT2CHRM1 SEQ ID NOS:9 and 21, MEST SEQ ID NOS:5 and 17, RNR1 SEQ ID NOS:10 and 22, CYP27B1 SEQ ID NOS:11 and 23 and ICAM1 SEQ ID NOS:12 and 24.

Example 3 Additional Aspects Provide for Diagnosis and/or Prognosis of Testicular Cancer, Comprising Use of the Biomarkers Disclosed Herein

Overview. Testicular germ cell tumors are the most common cancer of young men. Platinum-based chemotherapeutic agents improved survival of these tumors dramatically in the 1970's, to nearly 80 percent; in response to this success, etiologic and preventive research waned. However, in the intervening decades, incidence has doubled, and it has become clear that patients cured of germ cell tumors have serious and sometimes fatal sequelae. One of the few demonstrated risk factors is primary infertility (preceding cancer diagnosis), and age-period-cohort analyses implicate events during embryogenesis or early childhood (reviewed in Cortessis 2003).

Germ cells have been called the “ultimate” stem cells because they can give rise to all cell types of the body. Germ cells and their transformed derivatives share many properties with

embryonic stem cells, including expression of OCT4 and Nanog transcription factors (Sperger et al 2003) required for maintenance of stem cell pluripotency (Boyer 2005), capacity for both differentiation and transformation during in vitro manipulation, and gain of supernumerary material of common regions of chromosomes 12 and 17 (Draper 2004).

The mammalian germ line undergoes extensive epigenetic reprogramming during development. Widespread erasure of DNA methylation occurs in primordial germ cells as they migrate to the gonadal ridges (FIGS. 4 a and b). After the testes form, de novo DNA methylation (FIG. 4 b) establishes male-specific DNA methylation during germ cell maturation and spermatogenesis (Hajkova 2002). Consistent with the possibility that disruption of epigenetic reprogramming of the germ line leads to abnormal spermatogenesis and compromised male fertility, we (Houshdaran et al 2007) and others (Marques 2008) demonstrated DNA hypermethylation in sperm of men with poor quality sperm, a clinical indicator of sub-fertility.

According to the cancer stem cell hypothesis of carcinogenesis, malignant tumors include cells with the stem cell properties of self-renewal and pluripotency. This hypothesis addresses demonstrated features of stem cells and tumors, accounts in part for the transformed phenotypes and evolving behavior of malignancies, and largely explains the distribution of malignant potential among the tissue types of the body.

According to particular aspects, if the general hypothesis is correct, the rapidly emerging principles and tools of stem cell biology will likely have tremendous potential to shed light on specific mechanisms of carcinogenesis, and to provide molecular strategies for cancer prevention and treatment. However, research addressing the hypothesis requires the identification of stem cells within the tumor, an objective not yet attained for most human malignancies. Fortunately, this obstacle does not exist for germ cell tumors, for which the stem cell component is the germ cells themselves, which are readily identified.

As discussed herein, epigenetic change is a feature shared by normally differentiating stem cells and abnormally evolving cancer cells. The germ cell lineage is extensively reprogrammed during embryonic development, and the epigenetic state of germ cells is particularly susceptible to disruption during this time. Epidemiologic studies have consistently identified this same period as critical in germ cell carcinogenesis, and shown that infertility is associated with risk of germ cell tumors.

According to additional aspects of the present invention, disruption of epigenetic programming of germ cells during early development leads to both functional deficits manifest as sub-fertility in adulthood and increased malignant potential.

As described herein under Applicants' Examples 1 and 2, we first examined DNA methylation of sperm provided by men with poor fertility—a more common condition for which samples could be readily obtained. Our results disclosed herein are consistent with Applicants' additional conception, as they can be explained by disruption of the widespread erasure of DNA methylation that occurs early in the development of germ cells (see FIG. 4). FIGS. 4 a, b and c schematically depict mammalian primordial germ line development, showing that primordial germ cells undergo extensive epigenetic reprogramming during development, where widespread erasure of DNA methylation occurs in primordial germ cells as they migrate to the gonadal ridges). After the testes form, de novo DNA methylation establishes male-specific DNA methylation during germ cell maturation and spermatogenesis (see FIGS. 4 a and b). According to particular aspects of the present invention, DNA methylation of sperm from men with poor fertility is consistent with disruption of the widespread erasure of DNA methylation that occurs early in the development of germ cells (see FIG. 4 c).

DNA methylation analyses revealing epigenetic stem cell signature in epithelial cancers. In stem cells, polycomb group proteins reversibly repress genes required for differentiation. Using DNA methylation analysis of gene promoter sequences, polycomb target genes were recently found to be over-represented among genes that are silenced in cancers of the colon, ovary and breast by DNA hypermethylation of promoter sequences (Widschwendter et al 2007).

According to additional aspects of the present invention, the epigenetic state of stem cell components of human germ cell tumors and sub-fertile spermatozoa are compared with that of normal germ cell populations from which they originate to provide a set of biological markers for germ cell tumors and sub-fertile spermatozoa.

Materials and Methods:

Characterization of DNA methylation of cells representing very early and final stages of the male germ cell lineage (germ cell precursors and spermatozoa) identifying profiles that distinguish between these stages. Using an Illumina Infinium assay a quantitative assessment of DNA methylation is performed at over 25,000 DNA sequences in order to determine normal DNA methylation profiles in each cell type. By statistical analysis the sequences at which DNA methylation best distinguishes the epigenetic state of early versus late stages of normally developing male germ cells are identified.

Development and validation of MethyLight assays of DNA methylation at identified sequences. MethyLight assays are first be used to measure DNA methylation in the cell types used above, then to assess any cell types representing intermediate stages of development (e.g. gonocytes) for which fresh cells of sufficient quantity could not be. On the basis of these results, subsets of reactions representing probable stages of germ line development will be identified.

Assessment of DNA methylation of germ cells provided by men diagnosed with germ cell tumors and sub-fertility using the developed MethyLight assays. DNA of spermatozoa collected prior to treatment is assessed for patients diagnosed with both germ cell tumors and male-factor infertility. For germ cell tumor patients, germ cells taken from testicular tissue are also examined. For the set of patients for whom fresh tissue is collected at orchiectomy, germ cells from both tumor and normal regions of the testis are analyzed, while germ cell-rich tumors are analyzed, only, of specimens obtained as formalin-fixed paraffinembedded (FFPE) tissue. Use of FFPE tissue allows inclusion of a representative sample of histologic types of germ cell tumors, while analysis of the fresh tissue allows for separate examination of normal and transformed germ cells of the same patient. The resulting patterns are compared to those identified above, to confirm that aberrant epigenetic state of germ cell DNA is a component of the etiology of these conditions. These additional aspects comprise combining the knowledge and methods of cancer epidemiology, stem cell biology and epigenetics.

Etiologic studies of testicular germ cell tumors. Applicants have informed consent access to a collection of fresh testicular germ cell tumor tissue.

In vitro preparation of germ cell precursors. Human embryonic stem (ES) cells are propagated on mouse embryo fibroblast feeder cells as described (Reubinoff et al., 2000). Embryonal carcinoma cell lines are propagated as described previously (Pera et al., 1989). A multistep induction procedure incorporating several previously described protocols is used to convert ES cells into primordial germ cells at high efficiency. ES cells are treated with bone morphogenetic protein-2 for a brief 24 period in combination with activin and FGF-2 in chemically defined medium. After 24 hours the BMP-2 is removed and retinoic acid is added. A range of doses of each factor is employed in a matrix design over a variable time course to optimize the yield of c-kit positive/placental alkaline phosphatase positive cells. These cells are isolated by flow cytometry and subjected to Q-RTPCR to analyze for the presence of primordial germ cell and gonocyte specific genes such as VASA. Applicants estimate that up to 10% of cells will be vasa positive following optimal treatment.

Samples. Human ES and embryonal carcinoma cells are already available. Fresh testicular tissue representing normal embryonic, fetal, pre-pubescent and adult stages of development are available through an ongoing protocol of tissue collection at postmortem exam. Fresh testicular tissue from men diagnosed with germ cell tumors is collected at orchiectomy and made available. Paraffin-embedded formalin-fixed (PEFF) tumor blocks are obtainable from the discard repository of cancer surveillance programs and by requesting blocks to be prepared from tissue resected from patients who have provided consent for same. Normal sperm is taken from donated sperm bank samples and maintained in a research repository. Semen samples are collected by patients newly diagnosed with testicular germ cell tumors and infertility before treatment for either condition. To obtain semen samples from testis cancer patients, logistical procedures are used before initial treatment of testis cancer. Semen samples from infertility patients are available.

Sample processing. Primordial germ cells and gonocytes are isolated from embryonic and fetal gonads by the use of c-kit and placental alkaline phosphatase in combination with flow cytometry, following collagenase and Tryple Express digestion of the tissue. This protocol is evaluated for isolation of more mature germ cells and adapted by use of additional antibodies as required. Sperm is separated from seminal plasma as described (Houshdaran et al 2007). DNA is isolated from all cell types to be studied, and subjected to bisulfite conversion prior to analysis of DNA methylation.

Analysis of DNA methylation. Illumina Infinium Methylation assays are used, and which can generate DNA methylation data for 27,578 loci on sets of 12 samples at once, performing optimally using DNA isolated from fresh or fresh-frozen cells. The Infinium assay is used for samples for which abundant DNA can be isolated from fresh cells, and MethyLight assays for all other (fewer cells from fresh and all from FFPE tissue).

Statistical analysis. Analysis relating patterns of DNA methylation to each cell type and disease outcome is performed in collaboration with a mathematical statistician who specializes in statistical methods for analysis of DNA methylation data. Approximately 20 loci are identified for which DNA methylation levels, in combination, provide the highest sensitivity and specificity for distinguishing early from late cell types in the germ cell lineage. MethyLight assays are developed for these sequences, and DNA methylation profiles from germ cells of men with these conditions are compared systematically to the profiles of normal cells representing early and late stages of germ cell maturation using multivariate analysis implemented using machine learning algorithms.

Translational Potential. This Example provides a new combination of cancer epidemiology, stem cell biology, and epigenetics, and addresses multiple conditions ranging from infertility to several cancers.

Results. Abnormal DNA methylation of poor quality sperm. As described elsewhere herein, Applicants have identified a pattern of elevated DNA methylation in DNA of poor quality sperm (Houshdaran et al 2007). Notable features of the pattern are the association with a large proportion of interrogated sequences in multiple epigenetic contexts including promoter CpG islands of expressed genes, differentially methylated sequences of imprinted genes, and a repeated element, and a consistent dose response pattern of higher DNA methylation among samples with poorer values of each parameter of the standard semen analysis (see boxplots relating summary “M index” of MethyLight results at all associated sequences to individual semen parameters, FIG. 2). Of particular relevance to the subject matter of this Example were associations with sequences specific to cancer-related HRAS1 (p=0.00006), and NTF3 (p=0.00029) implicated in germ cell carcinogenesis based on protein function and presence in the region of chromosome 12p that is consistently amplified in malignant transformation of germ cells and embryonic stem cells.

Additional aspects, therefore, provide methods for diagnosis or prognosis of testicular or male germ-cell derived cancer, comprising: obtaining a sample of human sperm DNA from a test subject; determining, using the genomic DNA of the sample, the methylation status of at least one CpG dinucleotide sequence of at least one gene sequence selected from the group consisting of HRAS, NTF3, MT1A, PAX8, DIRAS3, PLAGL1, SFN, SAT2CHRM1, MEST, RNR1, CYP27B1 and ICAM1; and determining, based on the methylation status of the at least one CpG sequence, diagnosis or prognosis of testicular or male germ-cell derived cancer with respect to the test subject. In certain aspects, the determined methylation status of the at least one CpG sequence is hypermethylation. In particular aspects, determining the methylation status of at least one CpG dinucleotide sequence comprises treating the genomic DNA, or a fragment thereof, with one or more reagents to convert 5-position unmethylated cytosine bases to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties. In certain embodiments, treating comprises use of bisulfite treatment of the DNA. In particular aspects, the at least one gene sequence is selected from the group consisting of HRAS SEQ ID NOS:63 and 20, NTF3 SEQ ID NOS:2 and 14, MT1A SEQ ID NOS:4 and 16, PAX8 SEQ ID NOS:1 and 13, DIRAS3 SEQ ID NOS:3 and 15, PLAGL1 SEQ ID NOS:7 and 19, SFN SEQ ID NOS:6 and 18, SAT2CHRM1 SEQ ID NOS:9 and 21, MEST SEQ ID NOS:5 and 17, RNR1 SEQ ID NOS:10 and 22, CYP27B1 SEQ ID NOS:11 and 23 and ICAM1 SEQ ID NOS:12 and 24.

In certain aspects, the diagnosis or prognosis is of germ-cell derived testicular cancer.

Particular embodiments of the cancer diagnostic or prognostic assays comprise determining, using the genomic DNA of the sample, the methylation status of at least one CpG dinucleotide sequence of at least one gene sequence selected from the group consisting of HRAS, NTF3, MT1A, PAX8 and PLAGL1. In certain aspects, the at least one gene sequence is selected from the group consisting of HRAS SEQ ID NOS:63 and 20, NTF3 SEQ ID NOS:2 and 14, MT1A SEQ ID NOS:4 and 16, PAX8 SEQ ID NOS:1 and 13, and PLAGL1 SEQ ID NOS:7 and 19.'

Additional aspects provide a method for diagnosis or prognosis of testicular or male germ-cell derived cancer, comprising: obtaining a sample of human sperm DNA from a test subject; determining, using the genomic DNA of the sample, the methylation status of at least one CpG dinucleotide sequence of at least one gene sequence from each of a repetitive DNA element sequence group, a maternally imprinted gene sequence group, and a non-imprinted gene sequence group; and determining, based on the methylation status of the at least one CpG sequence from each of the groups, diagnosis or prognosis of testicular or male germ-cell derived cancer with respect to the test subject. In certain aspects, the determined methylation status of the at least one CpG sequence is hypermethylation. In particular implementations, the methylation status of at least one CpG dinucleotide sequence comprises treating the genomic DNA, or a fragment thereof, with one or more reagents to convert 5-position unmethylated cytosine bases to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties. In certain embodiments, treating comprises use of bisulfite treatment of the DNA. In certain aspects, the at least one gene sequence from a repetitive element group comprises at least one selected from the group consisting of SAT2CHRM1 SEQ ID NOS:9 and 21. In particular aspects, the at least one gene sequence from a maternally imprinted gene group comprises at least one selected from the group consisting of PLAGL1 SEQ ID NOS:7 and 19, MEST SEQ ID NOS:5 and 17, and DIRAS3 SEQ ID NOS:3 and 15. In certain embodiments, the at least one gene sequence from a non-imprinted gene group comprises at least one selected from the group consisting of HRAS SEQ ID NOS:63 and 20, NTF3 SEQ ID NOS:2 and 14, MT1A SEQ ID NOS:4 and 16, PAX8 SEQ ID NOS:1 and 13, SFN SEQ ID NOS:6 and 18, RNR1 SEQ ID NOS:10 and 22, CYP27B1 SEQ ID NOS:11 and 23 and ICAM1 SEQ ID NOS:12 and 24.

Additionally provided are methods for screening for agents that cause testicular or male germ-cell derived cancer, comprising: obtaining human ES-cell derived primordial germ cells; contacting the germ cells or descendants thereof, with at least one test agent; culturing the contacted germ cells or the descendants thereof under conditions suitable for germ cell proliferation or development; obtaining a sample of genomic DNA from the contacted cultured germ cells or the descendants thereof; determining, using the genomic DNA of the sample, the methylation status of at least one CpG dinucleotide sequence of at least one gene sequence selected from the group consisting of HRAS, NTF3, MT1A, PAX8, DIRAS3, PLAGL1, SFN, SAT2CHRM1, MEST, RNR1, CYP27B1 and ICAM1; and identifying, based on the methylation status of the at least one CpG sequence, at least one test agent that causes testicular or male germ-cell derived cancer. In certain aspects, the determined methylation status of the at least one CpG sequence is hypermethylation. In certain aspectgs, determining the methylation status of at least one CpG dinucleotide sequence comprises treating the genomic DNA, or a fragment thereof, with one or more reagents to convert 5-position unmethylated cytosine bases to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties. In certain embodiments, treating comprises use of bisulfite treatment of the DNA. In particular embodiments, the at least one gene sequence is selected from the group consisting of HRAS SEQ ID NOS:63 and 20, NTF3 SEQ ID NOS:2 and 14, MT1A SEQ ID NOS:4 and 16, PAX8 SEQ ID NOS:1 and 13, DIRAS3 SEQ ID NOS:3 and 15, PLAGL1 SEQ ID NOS:7 and 19, SFN SEQ ID NOS:6 and 18, SAT2CHRM1 SEQ ID NOS:9 and 21, MEST SEQ ID NOS:5 and 17, RNR1 SEQ ID NOS:10 and 22, CYP27B1 SEQ ID NOS:11 and 23 and ICAM1 SEQ ID NOS:12 and 24. In certain aspects, the at least one gene sequence is selected from the group consisting of HRAS SEQ ID NOS:63 and 20, NTF3 SEQ ID NOS:2 and 14, MT1A SEQ ID NOS:4 and 16, PAX8 SEQ ID NOS:1 and 13, and PLAGL1 SEQ ID NOS:7 and 19.

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1. A method for diagnosis or prognosis of testicular or male germ-cell derived cancer, comprising: obtaining a sample of human sperm DNA from a test subject; determining, using the genomic DNA of the sample, the methylation status of at least one CpG dinucleotide sequence of at least one gene sequence selected from the group consisting of HRAS, NTF3, MT1A, PAX8, DIRAS3, PLAGL1, SFN, SAT2CHRM1, MEST, RNR1, CYP27B1 and ICAM1; and determining, based on the methylation status of the at least one CpG sequence, diagnosis or prognosis of testicular or male germ-cell derived cancer with respect to the test subject.
 2. The method of claim 1, wherein the determined methylation status of the at least one CpG sequence is hypermethylation.
 3. The method of claim 1, wherein determining the methylation status of at least one CpG dinucleotide sequence comprises treating the genomic DNA, or a fragment thereof, with one or more reagents to convert 5-position unmethylated cytosine bases to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties.
 4. The method of claim 3, wherein treating comprises use of bisulfite treatment of the DNA.
 5. The method of claim 1, wherein the at least one gene sequence is selected from the group consisting of HRAS SEQ ID NOS:63 and 20, NTF3 SEQ ID NOS:2 and 14, MT1A SEQ ID NOS:4 and 16, PAX8 SEQ ID NOS:1 and 13, DIRAS3 SEQ ID NOS:3 and 15, PLAGL1 SEQ ID NOS:7 and 19, SFN SEQ ID NOS:6 and 18, SAT2CHRM1 SEQ ID NOS:9 and 21, MEST SEQ ID NOS:5 and 17, RNR1 SEQ ID NOS:10 and 22, CYP27B1 SEQ ID NOS:11 and 23 and ICAM1 SEQ ID NOS:12 and
 24. 6. The method of claim 1, wherein the diagnosis or prognosis is of germ-cell derived testicular cancer.
 7. The method of claim 5, comprising determining, using the genomic DNA of the sample, the methylation status of at least one CpG dinucleotide sequence of at least one gene sequence selected from the group consisting of HRAS, NTF3, MT1A, PAX8 and PLAGL1.
 8. The method of claim 7, wherein the at least one gene sequence is selected from the group consisting of HRAS SEQ ID NOS:63 and 20, NTF3 SEQ ID NOS:2 and 14, MT1A SEQ ID NOS:4 and 16, PAX8 SEQ ID NOS:1 and 13, and PLAGL1 SEQ ID NOS:7 and
 19. 9. A method for diagnosis or prognosis of testicular or male germ-cell derived cancer, comprising: obtaining a sample of human sperm DNA from a test subject; determining, using the genomic DNA of the sample, the methylation status of at least one CpG dinucleotide sequence of at least one gene sequence from each of a repetitive DNA element sequence group, a maternally imprinted gene sequence group, and a non-imprinted gene sequence group; and determining, based on the methylation status of the at least one CpG sequence from each of the groups, diagnosis or prognosis of testicular or male germ-cell derived cancer with respect to the test subject.
 10. The method of claim 9, wherein the determined methylation status of the at least one CpG sequence is hypermethylation.
 11. The method of claim 9, wherein determining the methylation status of at least one CpG dinucleotide sequence comprises treating the genomic DNA, or a fragment thereof, with one or more reagents to convert 5-position unmethylated cytosine bases to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties.
 12. The method of claim 11, wherein treating comprises use of bisulfite treatment of the DNA.
 13. The method of claim 9, wherein the at least one gene sequence from a repetitive element group comprises at least one selected from the group consisting of SAT2CHRM1 SEQ ID NOS:9 and
 21. 14. The method of claim 9, wherein the at least one gene sequence from a maternally imprinted gene group comprises at least one selected from the group consisting of PLAGL1 SEQ ID NOS:7 and 19, MEST SEQ ID NOS:5 and 17, and DIRAS3 SEQ ID NOS:3 and
 15. 15. The method of claim 9, wherein the at least one gene sequence from a non-imprinted gene group comprises at least one selected from the group consisting of HRAS SEQ ID NOS:63 and 20, NTF3 SEQ ID NOS:2 and 14, MT1A SEQ ID NOS:4 and 16, PAX8 SEQ ID NOS:1 and 13, SFN SEQ ID NOS:6 and 18, RNR1 SEQ ID NOS:10 and 22, CYP27B1 SEQ ID NOS:11 and 23 and ICAM1 SEQ ID NOS:12 and
 24. 16. A method for screening for agents that cause testicular or male germ-cell derived cancer, comprising: obtaining human ES-cell derived primordial germ cells; contacting the germ cells or descendants thereof, with at least one test agent; culturing the contacted germ cells or the descendants thereof under conditions suitable for germ cell proliferation or development; obtaining a sample of genomic DNA from the contacted cultured germ cells or the descendants thereof; determining, using the genomic DNA of the sample, the methylation status of at least one CpG dinucleotide sequence of at least one gene sequence selected from the group consisting of HRAS, NTF3, MT1A, PAX8, DIRAS3, PLAGL1, SFN, SAT2CHRM1, MEST, RNR1, CYP27B1 and ICAM1; and identifying, based on the methylation status of the at least one CpG sequence, at least one test agent that causes testicular or male germ-cell derived cancer.
 17. The method of claim 16, wherein the determined methylation status of the at least one CpG sequence is hypermethylation.
 18. The method of claim 16, wherein determining the methylation status of at least one CpG dinucleotide sequence comprises treating the genomic DNA, or a fragment thereof, with one or more reagents to convert 5-position unmethylated cytosine bases to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties.
 19. The method of claim 18, wherein treating comprises use of bisulfite treatment of the DNA.
 20. The method of claim 16, wherein the at least one gene sequence is selected from the group consisting of HRAS SEQ ID NOS:63 and 20, NTF3 SEQ ID NOS:2 and 14, MT1A SEQ ID NOS:4 and 16, PAX8 SEQ ID NOS:1 and 13, DIRAS3 SEQ ID NOS:3 and 15, PLAGL1 SEQ ID NOS:7 and 19, SFN SEQ ID NOS:6 and 18, SAT2CHRM1 SEQ ID NOS:9 and 21, MEST SEQ ID NOS:5 and 17, RNR1 SEQ ID NOS:10 and 22, CYP27B1 SEQ ID NOS:11 and 23 and ICAM1 SEQ ID NOS:12 and
 24. 21. The method of claim 20, wherein the at least one gene sequence is selected from the group consisting of HRAS SEQ ID NOS:63 and 20, NTF3 SEQ ID NOS:2 and 14, MT1A SEQ ID NOS:4 and 16, PAX8 SEQ ID NOS:1 and 13, and PLAGL1 SEQ ID NOS:7 and
 19. 